Article

pubs.acs.org/JAFC

Structures of Degradation Products and Degradation Pathways of

Aflatoxin B1 by High-Voltage Atmospheric Cold Plasma (HVACP)
Treatment
Hu Shi,† Bruce Cooper,‡ Richard L. Stroshine,† Klein E. Ileleji,*,† and Kevin M. Keener§
†
Department of Agricultural and Biological Engineering, ‡Bindley Bioscience Center, Purdue University, West Lafayette,
Indiana 47907-2093, United States
§
Department of Food Science and Human Nutrition, Iowa State University, Ames, Iowa 50011-1061, United States
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ABSTRACT: High-voltage atmospheric cold plasma (HVACP) is a novel nonthermal decontamination technology that has
potential for use in the food industry. In this study, HVACP was applied to treat pure aflatoxin B1 (AFB1) powder on a glass
slide. AFB1 was degraded by 76% using a 5 min HVACP treatment in air having 40% relative humidity. The degradation prod-
ucts of AFB1 were separated, and their molecular formulas were elucidated using liquid-chromatography time-of-flight mass
spectrometry (HPLC−TOF-MS). Six main degradation products were observed. The structures of the degradation products
were further clarified via orbitrap mass spectrometry by means of fragmentation of the parental ions. Two degradation pathways
were proposed on the basis of the structure of the degradation products. Among the six degradation products, two were
ozonolysis products of AFB1. The appearance of the other four degradation products indicates that AFB1 was degraded by other
reactive species besides ozone that were generated during HVACP treatment. Reactive oxygen gas species are suggested as the
major agents for aflatoxin degradation during HVACP treatment. Two degradation pathways of AFB1 by HVACP treatment were
proposed. One pathway involves reactions in which H•, OH•, CHO• radicals are added. The other involves epoxidation by HO2•
radicals and oxidation of AFB1by the combined effects of the oxidative species OH•, H2O2, and O3. According to the structure−
bioactivity relationship of AFB1, the bioactivity of the AFB1 samples subjected to HVACP treatment is significantly reduced
because of the disappearance of the C8C9 double bond in the furofuran ring in all of the major degradation products as well as
the modification of the lactone ring, cyclopentanone, and the methoxyl group.
KEYWORDS: aflatoxin B1, high-voltage cold plasma (HVACP), degradation products, orbitrap

■ INTRODUCTION
Aflatoxins are mycotoxins produced by the fungal species
A. (Aspergillus) flavus, A. parasiticus, and A. notimus.1 Aflatoxins
are highly toxic, immunosuppressive, mutagenic, and carcino-
genic to humans and animals.2,3 Among the aflatoxins that
are found, aflatoxin B1 (AFB1) is the most potent teratogen,
mutagen, and heptocarcinogen. It has been identified as a class 1
carcinogen by the International Agency for Research on Cancer.4
Varieties of agricultural products, such as corn, peanuts,
pistachios, and cottonseed are susceptible to aflatoxin con-
tamination. Prevention of contamination by toxigenic fungi is the
most rational and cost-effective approach to reducing the risks
associated with the presence of aflatoxin. However, it is not
always possible with current agronomic and storage practices,
especially when the environmental conditions are favorable
for the growth of toxigenic fungi.5 Therefore, detoxification has
gained importance in order to salvage aflatoxin-contaminated
grains and safeguard the grain industry from food safety concerns
and economic losses. Detoxification methods include cleaning
and sorting, use of food additives, treatment with ozone, and
treatment with electrolyzed oxidizing water.5−8
Lately, much attention has been paid to cold (nonthermal)
plasma as a novel microbial decontamination technology for the
food industry. It has the advantages of high efficiency and short
treatment time, it leaves no residue, and it has a low overall
impact on the quality of treated food products.9−11 In addition to
microbial degradation, the effectiveness of cold plasma tech-
nology in degradation of mycotoxins was recently studied by
investigators using different approaches of plasma generation.
It was reported that, in a model system, the mycotoxins AFB1,
DON, and NIV were successfully removed by 5 s of treatment
with microwave-induced argon plasma.12 Nitrogen gas plasma
generated by a static induction thyristor efficiently degraded
AFB1 to one-tenth of its original level within 15 min.13 Low-
temperature radio-frequency plasma degraded 88% of AFB1
within 10 min.14 In another study, multiple mycotoxins produced
by Fusarium, Aspergillus, and Alternaria species were effectively
degraded by high-voltage atmospheric cold plasma (HVACP)
treatment.15 HVACP is a cold plasma technology that generates
cold plasma by means of dielectric barrier discharge (DBD).
As opposed to other types of cold plasma technology, HVACP
treats the samples at atmospheric pressure thereby eliminating
the need for vacuum equipment. In addition, HVACP has the
advantage of high adaptability. Different geometries and varieties
of gases can be applied in conjunction with the treatment.10
HVACP treatment of aflatoxin has been shown to be highly
efficient in treatment of hazelnuts where 70% of the total
Received: April 6, 2017
Revised: June 17, 2017
Accepted: June 23, 2017
Published: June 23, 2017

© 2017 American Chemical Society 6222 DOI: 10.1021/acs.jafc.7b01604

J. Agric. Food Chem. 2017, 65, 6222−6230
Journal of Agricultural and Food Chemistry
aflatoxin was degraded by 12 min of HVACP treatment.16
In previous work by several of the authors of this paper, 10 min of
HVACP treatment reduced the aflatoxin level in a sample of corn
by 82%.17 These results indicate that HVACP is a very promising
technology for degrading mycotoxins. However, none of these
studies on HVACP treatment of aflatoxin included identification
of the degradation products, and the potential toxicity of the
degradation products was not discussed. Thus, there is a lack
of understanding of the reaction mechanisms involved with
HVACP treatment.15−17 This information is essential for a
more in-depth understanding of the HVACP process, and this
information is the basis for developing future applications.
Clarification of the reaction mechanism of HVACP treatment is
difficult, as hundreds of reactive species can be generated during
the HVACP process including electrons, radicals, ions, ozone,
and other reactive oxygen or nitrogen species.18 Thus, this study
is a continuation of previous work on treating aflatoxin-
contaminated corn using HVACP.17 Its purposes are to clarify
the structures of AFB1 degradation products, to elucidate the
reaction mechanism and degradation pathway of AFB1, and to
identify reactive gas species that are responsible for AFB1
degradation. The toxicity of the degradation products is also
discussed on the basis of their structures.
In this study, pure AFB1 powder was placed on a glass slide
and was treated using HVACP. The degradation products
were separated, and their chemical formulas were elucidated
using liquid-chromatography time-of-flight mass spectrometry
(HPLC−TOF-MS), which has previously been used as an
effective tool in analyzing aflatoxin degradation products.19−21
The structures of the reaction products were further investigated
using orbitrap mass spectrometry. This is a new technology used
for high-resolution mass spectrometry and has the features of
miniature design, high-speed detection, and excellent quantifi-
cation. It can be used for both reaction product identification and
molecular structure characterization.22,23
■ MATERIALS AND METHODS
Chemicals and Reagents. Aflatoxin B1 (2,3,6a,9a-tetrahydro-4-
methoxy-cyclopenta[c]furo[2′,3′,4,5]furo[2,3-h]chromene-1,11-dione;
C17H12O6, purity >98%) was purchased from Cayman Chemicals Inc.
(Ann Arbor, MI, U.S.A.). Chloroform and 200-proof ethanol were
obtained from the campus laboratory store. AFB1 powder was dissolved
in chloroform and serially diluted to a concentration of 50 μg/mL, and
the AFB1 standard solution was stored at −5 °C in a freezer prior to
being used in the tests.
Treating AFB1 with HVACP. Figure 1 is a schematic of the
experimental set-up for HVACP treatment of AFB1 which was placed on
a glass slide. Standard AFB1 solution in chloroform (100 μL; 50 μg/mL)
was pipetted onto a glass slide. This was followed by a wait time of 2 h to
allow the chloroform to fully evaporate. Thus, about 5 μg of aflatoxin in
powder form was subjected to HVACP treatment. The glass slide was
Figure 1. Schematic of experimental setup for HVACP treatment of
aflatoxin in corn.
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placed inside a translucent polypropylene box (Grainger Inc., Lake
Forest, IL, U.S.A.). Boxes were sealed inside a high-barrier Cryovac
B2630 film in order to prevent leakage of the filled gas as well as to
contain the gas species that were generated. The air used as a fill gas
(78% N2, 22% O2) was purchased from a local gas supplier and had a
certificate of analysis. The gas in the tank had a relative humidity (RH) of
5%. In order to increase the RH to 40%, the working gas was passed
through a water bubbler. The gas flow rate and water depth were
adjusted, and the humidity of the exiting air was measured with a
psychrometer (Extech Instruments Inc., Nashua, NH, U.S.A.). The final
RH of the humidified air was 40 ± 3%. The storage bags containing
AFB1 on the glass slide were filled with the working gas and were purged
three to five times for 2 min to ensure purity of the gas in the bag.
HVACP treatments were conducted utilizing the HVACP system
(Phenix Technolgies, Accident, MD, U.S.A.) shown in Figure 1. This is
patented technology developed by Dr. Keener while he was working at
Purdue University.24 The parameters for the experimental set-up are
shown in Table 1. The temperature of the plasma was measured using an
Table 1. Parameters Describing the HVACP System
parametera value
powder/frequency 200 W / 50 Hz
applied voltage 90 kV
bag size (L × W) 35.56 cm × 26.82 cm
compartment box size (L × W × H) 27.31 cm × 17.78 cm × 4.45 cm
gap distance 4.44 cm
gas temperature ≈40 °C
a
L: length, W: width, H: height.
infrared thermometer (Omega Engineering Inc., Bridgeport, NJ,
U.S.A.).The AFB1 samples were treated for 1, 2, or 5 min. For each
treatment time, the experiment was conducted in triplicate. After
treatment, the AFB1 samples were stored in their sealed bag at room
temperature for 24 h. This storage period allows the generated plasma to
decompose so that the gas in the bag is once again air. It has been
demonstrated that the majority of aflatoxin degradation occurs during
the HVACP treatment time rather than during the storage period.17
HPLC−MS Analysis. The HVACP-treated and untreated AFB1
samples were carefully rinsed multiple times with 1 mL of a 50% ethanol
aqueous solution to extract the AFB1 and degradation products.
The extracts then were transferred to an Eppendorf tube (2.0 mL) and
were stored at −5 °C in a freezer before being subjected to HPLC
analysis with mass spectrometry. HPLC−MS data on the AFB1 degra-
dation products were obtained on a time-of-flight (TOF) instrument
system (Agilent Technologies, Santa Clara, CA, U.S.A.) equipped with
an 1100 series binary solvent delivery system and an autosampler.
Chromatography was performed on a 2.1 × 150 mm Waters Xterra C18
column with a particle size of 3.5 μm. The injection volume was 10 μL,
and the flow rate was 300 μL/min. The mobile phase was a gradient
prepared from distilled water with 0.1% formic acid (component A) and
acetonitrile with 0.1% formic acid (component B). Gradient elution
started with 10% B for 1 min, after which B was increased linearly to 95%
in 20 min and subsequently kept isocratic for 1 min. The proportion
of B was then decreased to 10% in 1 min and kept isocratic for 7 min.
The total run time was 30 min. The MS was run with positive elec-
trospray ionization (ESI), and the data were collected over the range of
75−1000 m/z. High mass accuracy was ensured by infusing lock mass
calibrants corresponding to 121.0508 and 922.0098 m/z.
HPLC−MS−MS Analysis. The same chromatographic conditions
were used for the HPLC−MS−MS analysis as described in the HPLC−
MS section above. Tests were performed using a Thermo LTQ Orbitrap
XL mass spectrometer. The analysis used positive polarity electrospray
ionization. High mass accuracy fragmentation data were acquired
using the data-dependent scanning mode. Fourier transform-based
mass spectrometry (FTMS), with a resolution of 60 000 and a range of
50−1100 m/z was used for full scan analysis. An FTMS resolution of
7500 was used for MS−MS data acquisition in the collision induced
dissociation (CID) mode. The five most intense ions were acquired with
DOI: 10.1021/acs.jafc.7b01604
J. Agric. Food Chem. 2017, 65, 6222−6230
Journal of Agricultural and Food Chemistry
Figure 2. Chromatograms of AFB1 untreated (5 μg/mL) in 50% ethanol solution (A) and AFB1 sample treated by HVACP in ambient air for 5 min (B).
a minimum signal of 1000, an isolation width of 2, a normalized collision
energy of 35 eV, a default charge state of 1, an activation Q of 0.250, and
an activation time of 30 ms.
■ RESULTS AND DISCUSSION
Formation of AFB1 Degradation Products with HVACP
Treatment Time. Figure 2 shows the total ion chromatograms
of the untreated AFB1 sample and of a sample treated by HVACP
in ambient air for 5 min. Only one peak appeared for the AFB1
sample without HVACP treatment, whereas seven relatively
large peaks (including the AFB1 peak) were observed for the
HVACP treated sample. This is a good indication that the AFB1
was degraded by the HVACP treatment. Six major degradation
products are shown in Figure 2B. The retention time and peak
shape revealed satisfactory separation of the degradation
products except for products 4 and 5, whose retention times
were nearly the same so that their peaks were not completely
separated. Figure 3 shows the change in the response value for
AFB1 and degradation products (P1−P6) with increasing
HVACP treatment time in air. As treatment time increased,
AFB1 was gradually decomposed. All degradation products
gradually increased, except for degradation product P2, which
increased in the first 2 min of HVACP treatment and then
decreased with longer HVACP treatment time. The decrease in
P2 suggests that it might be an intermediate reaction product that
was further converted into other degradation products. The
coefficients of variance (COV) for measurements of AFB1 and
degradation products were between 7% and 14%. The reduction in
AFB1 level followed an exponential decay trend during the 5 min
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HVACP treatment, which is consistent with our previous
observation on the aflatoxin degradation kinetics in corn by
HVACP treatment.17 About 76% of the AFB1, as determined
from the areas of the peaks, was degraded after 5 min of HVACP
treatment.
Molecular Formulas of Degradation Products. As an aid
to identifying the molecular formulas of the degradation
products of AFB1, data for both AFB1 and its HVACP
degradation products are summarized in Table 2. The data
included were retention time, proposed formula, experimental
mass, mass error, double bond equivalent (DBE), and the score.
(The overall score ranges from 0 to 100%, with a score closer to
100% being better.) Compared with the theoretical mass
obtained from the proposed molecular formula, the mass
determined by TOF-MS experiments had less than a 6 ppm
error. The results showed that since the product masses were
accurately determined, the elemental composition could be
determined by considering all possible elemental compositions.
Because AFB1 was treated by HVACP in a pure system, AFB1
and its degradation products could only be composed of four
elements: carbon, oxygen, hydrogen, and nitrogen. The
molecular formulas were proposed on the basis of the products’
observed isotope distribution patterns and their exact mass by
using MassHunter Qualitative Analysis software (Agilent
Technologies, Santa Clara, CA, USA). As an example, the
molecular formulas and several possible molecular compositions
of product 2 were proposed as C17 H14 O7, C18H10N4O3, and
C16H8N7O2 with scores of 95, 89, and 90%, respectively. Since
C17 H14 O7 has the highest score, it is the most likely to be the
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Figure 3. Relative change in response value of AFB1 and degradation products 1 through 6 (P1−P6) for AFB1 samples with increasing HVACP
treatment time in air. (Note: AFB1 and P2 are normalized so that they can be plotted on the same scale.)

Table 2. Proposed Formulas for AFB1 and Its Degradation furofuran ring (products 1−6), the cyclopentenone (products 1, 6)
Products Obtained Using LC−TOF−MS and the methoxy group (product 1).
Reaction Mechanism and Degradation Pathway of
retention observed
proposed time proposed mass diffb score AFB1 by HVACP Treatment. There are three possible agents of
products (min) formula (m/z)a (ppm) DBEc (%) cold plasma treatment that could act on food-borne pathogens.
1 10.1 C16 H16 O6 305.1028 −1.98 9 98.12 They are generated heat, ultraviolet (UV) radiation, and reactive
2 11.0 C17H14O7 331.0820 −1.67 11 98.7 gas species.25 Since HVACP treatment is a nonthermal process,
3 11.5 C14 H12 O5 261.0772 −5.92 9 90.32 the temperature of the sample during HVACP treatment is
4 11.9 C14 H10 O6 275.2560 −3.62 10 96.24 around 40 °C, which is well below the temperature (ca. 260 °C)
5 12.0 C17 H12 O7 329.0666 −4.01 12 92.00 required for thermal decomposition of AFB1 in powder form.26
6 12.5 C19 H18 O8 375.1085 −2.93 11 96.09 Thus, the contribution of heat generated during HVACP
AFB1 13.4 C17 H12 O6 313.0716 −2.52 12 99.1 treatment is considered to have a negligible effect on aflatoxin
a
The m/z of [M + H]+. bDiff = mass difference. cDBE = double bond degradation. During HVACP treatment, UV light was emitted
equivalents. with peaks at 316, 337, and 357 nm due to the N2 species
transition.17 However, the emission intensity of UV light during
molecular formula. Another indicator is the double bond HVACP is less than 50 μW/cm2.27 This is much lower than the
equivalent (DBE) values of the degradation products, which UV intensity (1820−13 200 μW/cm2) required for effective
should be similar to that of AFB1. The AFB1 consists of 17 degradation of aflatoxin.28 Thus, the effect of UV emission is
carbon atoms and 12 hydrogen atoms with a DBE value also considered to be insignificant. This conclusion is further
of 12. The DBE values of the molecular formulas, C17H14O7, corroborated by the observation that the degradation products of
C18H10N4O3, C16H8N7O2 are 11, 16, and 14.5, respectively. Since HVACP treatment are different from the three identified major
the molecular formula and DBE of C17H14O7 are the most similar degradation products of AFB1 subjected to UV radiation.28 Thus,
to AFB1, it is the most likely of the three to be the molecular it is postulated that the reactive gas species generated during
formula. plasma treatment is responsible for aflatoxin degradation. For
Proposed Structures for Degradation Products. To HVACP treatment, when air was applied as the gas inside
elucidate the structure of the six degradation products of AFB1, the bag reactive oxygen and nitrogen species were generated,
the degradation products were further analyzed by orbitrap which can degrade food-borne pathogens, pesticides, allergens,
MS−MS to determine the exact masses of the fragmentation and other undesirable chemical compounds.29−32 The authors
ions. This enabled postulation of their most probable parental believe that the reactive oxygen species, rather than the nitro-
structure and the structure of the reaction products of AFB1. gen species, plays a major role in aflatoxin degradation during
On the basis of the accurate masses of the parent ions and HVACP treatment. There are two reasons. First, much lower
fragments obtained from MS−MS, the analysis of the structures degradation of aflatoxin was achieved in pure nitrogen gas than in
of the six degradation products are shown in Figure 4. The struc- air, and there are lower concentrations of ozone and other
tures of the AFB1 degradation products by HVACP are sum- oxidizing species generated in nitrogen gas than in air.17 The
marized in Figure 5. The structures of the six degradation prod- second reason is that we did not find the nitrogen molecule in
ucts (P1−P6) are similar to the structure of AFB1. The degra- the degradation products of AFB1 during HVACP treatment.
dation by HVACP resulted in the modification of the AFB1 The authors expected to see it in the products if it had played a
6225 DOI: 10.1021/acs.jafc.7b01604
J. Agric. Food Chem. 2017, 65, 6222−6230
Journal of Agricultural and Food Chemistry Article

Figure 4. Orbitrap MS−MS spectra and proposed fragmentation (insets) of degradation products of AFB1 by HVACP.

Figure 5. Proposed structure of degradation products (P1−P6) of AFB1 produced by HVACP treatment.

significant role in degrading aflatoxin. One of the major reactive
species generated by HVACP is ozone.33−35 It has been shown
that it effectively degrades aflatoxins in seeds, grains, and
foods.36−38 The effect of generated ozone on aflatoxin degradation
6226
during HVACP treatment has been supported by this study.
Two aflatoxin degradation products, product 1 (C16H16O6) and
product 2 (C17H14O7) have been shown to be major ozono-
lysis products of the treatment of AFB1 by aqueous ozone.19,20
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Journal of Agricultural and Food Chemistry
Four other new major degradation products were identified,
indicating that the degradation of AFB1 by HVACP treatment
involves new pathways and reactive oxygen gas species other than
ozone.
On the basis of the structure of the six degradation products
of AFB1 produced by HVACP treatment, two degradation
pathways were proposed as shown in Figure 6 and Figure 7.
Figure 6. First degradation pathway of AFB1 by HVACP treatment.
The first pathway is from AFB1 to degradation products
C17H15O7 (m/z 331.0821), C16H17O6 (m/z 305.1028), and
C19H19O8 (m/z 375.1056). The second pathway is from AFB1 to
degradation products C14H13O5 (m/z 261.0755), C14H11O6
(m/z 275.0549), and C17H13O7 (m/z 329.0666).
The first degradation pathway primarily involves addition
reactions in which a water molecule (H2O), hydrogen molecule
(H), or aldehyde group (CHO) is added to AFB1. The first
branch reaction starts with the addition of water molecules
(hydration reaction) to the C8C9 double bond at the furan
ring of AFB1 to form the degradation product C17H15O7
(m/z 331.0821). The methoxy group (−OCH3) of AFB1 was
cleaved to form the intermediate product C16H13O6 (m/z 301.0712),
and the carbonyl groups of the intermediate product C16H13O6 were
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Figure 7. Second degradation pathway of AFB1 by HVACP treatment.
further hydrogenated to form the degradation product C16H17O6
(m/z 305.1028). The second branch of the reaction is the addition of
an aldehyde group (CHO) to form the intermediate product,
C19H15O8 (m/z 371.0767). Next, the carbonyl groups in the
lactone ring and cyclopentanone of this intermediate product
were hydrogenated to form the degradation product, C19H19O8
(m/z 375.1056). From the first degradation pathway of AFB1, the
crucial reactive agents are the hydrogen atom (H) and the hydroxyl
radical (OH•), which were generated in the HVACP system by the
breakdown of water molecules.39 These two species are responsible
for hydration and hydrogenation to form new degradation products.
Another reactive agent generated by HVACP is the aldehyde
(CHO•) radical, which is formed in the HVACP system when carbon
dioxide (CO2) is present in the used gas.40
The second pathway mainly involves epoxidation and oxi-
dation reactions. The first branch is the formation of degradation
product C17H13O7 (m/z 329.0666) through epoxidation of
the terminal double bond of AFB1. The epoxidation reaction
could be attributed to the hydroperoxyl radical (HO2•) that is
generated during HVACP treatment. Its concentration increases
when the relative humidity of the air is higher.41 HO2• is one type
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of peroxyl radical that reacts with double bonds and leads to
epoxide formation.42 In the second branch, the furofuran ring of
AFB1 is cleaved, and the degradation product C14H11O5
(m/z 261.0755) is formed. Further oxidation of this product
leads to formation of another degradation product, C14H11O6
(m/z 275.0549). Several oxygen-containing reactive species,
such as atomic oxygen (O), the hydroxyl radical (OH•),
hydrogen peroxide (H2O2), and ozone (O3) have been found
to be present during HVACP treatment.27,35,43 Since the
concentration of these species during HVACP treatment was
not quantified in this study, it is unclear which specific species
reacted with AFB1. It is more likely the degradation of AFB1
is through the combined action of these species, since they
coexisted during the treatment and are interconvertible.
For example, H2O2 and OH• can be formed by decomposition
of O3 with a water molecule. Also, H2O2 can be formed by the
combining of two OH• groups. The specific contribution from
each species during HVACP treatment depends on the relative
humidity and the composition of the used gas. For HVACP
treatment in dry air, O3 is considered the major reactive species.
However, in humid air higher concentrations of OH• and H2O2
are generated.35,44,45 It has been reported that under humid
conditions or when in a liquid matrix, OH• and H2O2 are the
major reactive species that achieve decontamination of organic
compounds and microbes.32,45 In this study, the relative
humidity of air during HVACP treatment was at an intermediate
level (40%). Therefore, it is expected that degradation of AFB1
was through simultaneous reactions with OH•, H2O2, and O3.
In summary, the second pathway involves an epoxidation
reaction by HO2• radicals and oxidation reactions through the
combined effects of the oxidative species OH•, H2O2, and O3.
Since different plasma generation methods, such as micro-
wave-induced argon plasma, nitrogen-gas plasma by a static-
induction thyristor, radio-frequency cold plasma in air, and
HVACP, generate plasma with different profiles of reactive
species, their degradation of aflatoxin should involve different
reaction mechanisms. However, the degradation products and
reaction mechanisms of aflatoxin degradation associated with
microwave-induced argon plasma and with nitrogen plasma
generated by a static-induction thyristor have not yet been clari-
fied by the investigators in their studies.12,13 Thus, the authors of
this paper cannot compare the reaction mechanisms of these
plasma technologies to HVACP. Five major aflatoxin degrada-
tion products from radio-frequency cold plasma were identified.
They are all different from the degradation products by HVACP
treatment.14 A similarity to this study is that the radio-frequency
cold plasma was generated with air as the applied gas. The OH•
and H• radicals were generated and were also reported as major
reactive species for aflatoxin degradation by radio frequency.14
In order to make additional comparisons of the efficacy and
mechanisms of the different cold plasma technologies, the
profiles and concentrations of the generated gas species would
have to be determined using optical emission and absorption
spectroscopy.
Toxicity Analysis of the Degradation Products. Since
the discovery of aflatoxin in the early 1960s, the toxicology
of aflatoxins have been widely studied.46,47 The relationship of
structure-to-bioactivity (toxicity, carcinogenicity, and mutage-
nicity) of aflatoxins has been elucidated.48,49 AFB1 has an optimal
molecular structure for acute toxicity, mutagenicity, and carcino-
genicity. Any changes in the furofuran ring, the lactone ring, the
cyclopantenone, or the methoxyl structure would result in a
marked reduction in biological activity.50 The furofuran moiety
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of the aflatoxin structure is essential for toxic and carcinogenic
activities, and the double bond in the terminal furan ring is
a notably important determinant of toxic and carcinogenic
potency.49 As shown in Figure 5, all of the six proposed major
degradation products of AFB1 lost their double bond, and the
degradation products were different from AFB1 by further
modification of the furofuran ring (products 1−6), lactone ring
(products 1, 6), cyclopantenone (products 1, 6), or the methoxyl
structure (product 1). Therefore, on the basis of the relationships
between chemical structure and biological activity reported in the
literature, the authors predict that the toxicity, carcinogenicity,
and mutagenicity of the HVACP degradation products will be
substantially less than those attributes of AFB1. This has been
confirmed by earlier research on treating aflatoxin B1 with
gaseous and aqueous ozone. In those studies, the toxicity of the
AFB1 degradation products was greatly reduced or disappeared
for the modified structures.51,52 Biological tests of toxicity of
treated aflatoxin samples were not conducted in both previous
studies of HVACP treatment or in this study.15−17 For other
types of cold plasma technologies, including microwave-induced
argon plasma and radio-frequency-induced nitrogen plasma,
biological tests have demonstrated markedly reduced toxicity in
treated samples. This is a good indicator that the cold plasma
treatment will also reduce toxicity. Nevertheless, the authors
recommend that additional bioactivity tests be conducted in the
future to ensure that HVACP treated samples are safe for animals
and humans. Testing could be conducted using duckling, Ames,
or cell model tests.
■ AUTHOR INFORMATION
Corresponding Author
*K.E.I.: Phone: 765-494-1198; E-mail: ileleji@purdue.edu;
Fax: 765-496-1115.
ORCID
Hu Shi: 0000-0002-5577-931X
Klein E. Ileleji: 0000-0002-7377-9671
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This study was supported by a 2010 Team Award from the
NC-213 Anderson Research Grant Program. The project
was entitled Reduction of Mycotoxin Levels in Distillers Grains.
The authors are grateful for the assistance with the HVACP
system provided by Ms. Jean Jensen and Zifan Wan, research
scientists from Purdue’s Food Science Department.
■
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6230 DOI: 10.1021/acs.jafc.7b01604

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